One of the newest, most provocative hypotheses regarding the pathogenesis of ALS is the excitotoxic hypothesis. Glutamate, the primary excitatory neurotransmitter in the brain, can exert specific neurotoxic effects and has been implicated as a causal factor in certain chronic and acute neurological diseases. In this proposal, we intend to examine various aspects of glutamate regulation in ALS and to determine whether chronic abnormalities in glutamate metabolism can cause motor neuronal degeneration, as follows. (1) We will examine multiple aspects of glutamate metabolism which are associated with its transmitter function(including glutamate uptake, glutamate synthetase, glutaminase, and glutamate dehydrogenase), using postmortem spinal cord and brain from ALS patients. Significant, large increases in CSF glutamate have already been demonstrated. The advantage of this plan is that it includes measurements of multiple enzymes and physiologic processes in the same patient tissues, providing a correlated analysis of glutamate synthesis and catabolism in detail. (2) To explain how altered glutamate metabolism could cause selective motor neuron death in ALS, we will define the selective distribution of glutamate receptors in normal human spinal cord, using newly available probes for receptor subtypes. Glutamate toxicity mediated via non-NMDA receptors may occur in ALS. Understanding the cellular and molecular localization of the glutamate receptor protein may help in delineating the role of glutamate systems in ALS. (3) Finally, we propose to develop a model of chronic glutamate toxicity, using cultured organotypic spinal cord slices, to test the hypothesis of excitotoxic motor neuron damage, under controlled conditions. Creation of in vitro models allows for experimental control, pharmacologic manipulation, and testing of therapeutic options that is impossible in ALS patients. These experiments will test the overall hypothesis that disturbances in glutamate metabolism play an important pathophysiologic role in motor neuron death in ALS. If this is true, it would have fundamental consequences for our understanding of how the motor neuron works, what makes it selectively vulnerable in motor neuron diseases, and how to approach it neuropharmacologically.